EP1877794A1 - Immobilisation de carbohydrates antigéniques pour contribuer à la détection de micro-organismes pathogènes - Google Patents

Immobilisation de carbohydrates antigéniques pour contribuer à la détection de micro-organismes pathogènes

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Publication number
EP1877794A1
EP1877794A1 EP06733024A EP06733024A EP1877794A1 EP 1877794 A1 EP1877794 A1 EP 1877794A1 EP 06733024 A EP06733024 A EP 06733024A EP 06733024 A EP06733024 A EP 06733024A EP 1877794 A1 EP1877794 A1 EP 1877794A1
Authority
EP
European Patent Office
Prior art keywords
lps
salmonella
polysaccharide
carrier
sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP06733024A
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German (de)
English (en)
Inventor
Aldert Anthonie Bergwerff
Gertruda Cornelia Antonia Maria Bokken
Bertha Gerarda Maria Gortemaker
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
RNA Holding BV
Original Assignee
RNA Holding BV
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Filing date
Publication date
Application filed by RNA Holding BV filed Critical RNA Holding BV
Priority to EP06733024A priority Critical patent/EP1877794A1/fr
Publication of EP1877794A1 publication Critical patent/EP1877794A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • the invention relates to the field of chemistry and diagnosis, more in particular to diagnosis of current and/or past and/or symptomless infections or of a history of exposure to a gram-negative -bacterium (such as an enterobacteriaceae or a legionella). Even more in particular, the invention relates to the screening of animals or animal products for the presence of unwanted/undesired microorganisms.
  • the invention further relates to a method for screening samples for the presence of antibodies directed against unwanted/undesired microorganisms and preferably such a method is performed with help of a biosensor.
  • the invention also relates to a method for immobilising polysaccharides to solid surfaces.
  • the invention furthermore provides solid surfaces with immobilised polysaccharides as well as applications of such surfaces.
  • the world is full of gram-negative bacteria, many of which are members of the family Enterobacteriaceae. Members of this family are found in the gastrointestinal tract of animals, but many are also free living in soil and water. Members of the family Enterobacteriaceae have very complex antigenic structures. Moreover, they comprise multiple antigens that are identified as K antigens, H antigens and O antigens.
  • the K antigen is the acidic polysaccharide capsule. The capsule has many functions including evasion from the immune system of the infected host and adhesion to the epithelium of the host.
  • the H antigen is located on the flagella.
  • LPS lipopolysaccharides
  • LPS is composed of lipid A which is buried in the outer membrane, a short carbohydrate core and optionally a chain of polysaccharides that is made up of repeating units.
  • the O-antigens are located on the polysaccharide.
  • Lipid A is the toxic constituent of the LPS. As cells lyse, LPS is released, leading to fever and complement consumption. It also interferes with coagulation and at high concentrations eventually leads to a state of shock.
  • enterobacteriaceae salmonella
  • Salmonella enterica A large number of the subspecies of the genera of Salmonella enterica are important pathogenenic bacteria for humans and animals. Besides that animals go into a pathological episode, animals can be symptomless carriers of the bacteria. Contaminated animals can be a source of these pathogens threatening public health for example through the food that these animals produce. As many stakeholders consider the number of food-borne salmonella infections unacceptable, measures have to be taken to contain this pathogen in the food chain. Salmonella is of major significance as a pathogenic microorganism in food-borne infections in humans, causing mild to severe clinical effects. In The Netherlands, 5% of all identified cases of gastroenteritis is salmonellosis (Edel et al., 1993; Hoogenboom Verdegaal et al., 1994).
  • Detection of immunoglobulins in the body fluids of organisms is a way to establish a history of exposure of animals and humans to infectious agents.
  • a humoral response against salmonella antigens can be detected in chickens 1 week post-infection and persists for at least 10 weeks even if the bird is no longer culture-positive (Holt, 2000).
  • the antigenic determinants of salmonella are, as described above, composed of somatic (O), flagellar (H) and surface (Vi) antigens (Holt, 2000). Variations in the composition of antigens correlate with different salmonella serotypes.
  • serology is faster than culture-typing of the disease- causative organism.
  • Fast and specific detection of potential salmonella-positive herds and flocks is of importance in order to take adequate measures in production processes.
  • the detection of antibodies in serum and blood samples from food-producing animals reporting the presence of zoonotic pathogens is therefore of significance.
  • Such information is then used as the input for risk- assessment and rational slaughtering of potentially pathogen-contaminated animals in order to be able to increase food safety, but also to improve occupational hazards and to reduce spreading of the pathogens in the environment.
  • ELISA have most commonly been used (Barrow, 2000). Agglutination tests have been used successfully to eradicate Salmonella pullorum from poultry flocks. However, the approach is cumbersome, laborious and not suitable for large-scale screening programs according to modern standards. Several ELISA procedures, which are considered relatively cheap and fast, have therefore been developed to detect anti-S. enteritidis and S. typhimurium antigen responses in poultry sera (Barrow et al., 1996; Thorns et al., 1996; de Vries et al., 1998; Barrow, 2000; Yamane et al., 2000).
  • a biosensor is defined as an analytical device consisting of (i) a re-usable immobilized biological ligand that 'senses' the analyte, and (ii) a physical transducer, which translates this phenomenon into an electronic signal.
  • SPR surface plasmon resonance
  • the goal of the present invention is to provide for a method that has an improved sensitivity and/or an improved robustness.
  • This goal has been reached by developing a carrier with immobilised somatic or so-called O-antigens.
  • the O-antigens are located on the lipopolysaccharides and the composition of the polysaccharide varies and corresponds with the serovar of the salmonella (sub)species. Every serotype can, amongst others, be described by a number of O-antigens and are typically coded with a number, such as O4, 06 or 012.
  • the O-antigens can be found as repeating units on the polysaccharide part of the LPS.
  • the length of the polysaccharide also varies and can be between zero (rough LPS) and more than 50 repeating units (smooth LPS).
  • each group comprises at least one specific O-antigen.
  • the salmonella serovars of importance in chicken and pigs are listed with their O- antigen profile in Table 1. In Denmark, Germany, Greece and The Netherlands, S. typhimurium. Dependent of country, other important isolates from pigs were S. derby (17.1%), S. infantis (8.0%), S. panama (5.1%), S. ohio (4.9%), S. london (4.4%), S. li ⁇ ingstone (3.1%), S. ⁇ irchow (2.7%), S. bredeny (2.1%), S. mbandaka (1.1%), S. Brandenburg (1.0%), S. goldcoast (0.8%).
  • Salmonella Chicken (C)/ pigs O-antigen serogroup serovar (P) profile Salmonella Chicken (C)/ pigs O-antigen serogroup serovar (P) profile
  • the invention provides a method for immobilisation of a polysaccharide on a carrier, comprising contacting said polysaccharide with an oxidising agent and a polymer comprising at least two amine and/or amide groups to obtain a polysaccharide-polymer complex and coupling said polysaccharide-polymer complex to said carrier.
  • the polymer can be any polymer that contains at least two amine and/or amide groups.
  • Said at least two amine and/or amide groups preferably cross-link said polymer to said polysaccharide and said carrier.
  • said polymer comprises at least 4 and more preferably at least 7 amine or amide groups.
  • the polymer comprises at least 10 building blocks. Building blocks of a polymer share characteristic reactive groups that enable elongation of the polymer.
  • a preferred building block is an amino acid or a functional part, derivative and/or analogue thereof.
  • said polymer comprises a protein.
  • a protein comprises at least one polypeptide chain comprising at least 10 amino acids or functional equivalent thereof.
  • a protein contains at least constituents having free amine and/or amide groups.
  • the protein can also be a multimer comprising at least two polypeptide chains that are covalently or non-covalently linked to each other.
  • the protein may comprise modifications such as those common to biological systems such as post-translational glycosylation.
  • the protein may also be artificially modified or provided with a further group as long as it has the mentioned amine and/or amide groups available.
  • said polysaccharide is derived from a gram- negative bacterium.
  • the sensitivity of such a prepared carrier is much improved when the lipopolysaccharide (O-antigen) before the immobilisation on the carrier is oxidised in the presence of a polymer comprising at least two amine and/or amide groups, preferably a protein.
  • a polymer comprising at least two amine and/or amide groups, preferably a protein.
  • an activated carrier for example a sensorchip
  • the available aldehyde groups react with hydrazide to form hydrazon.
  • the following reduction stabilises not only the covalent binding between the carrier (for example a carrier comprising dextran) and the polysaccharide but also the imine binding between protein and polysaccharide.
  • polysaccharides (O antigens) of different salmonella sera types have been immobilised on a carrier.
  • the prepared carriers were subsequently subjected to an SPR-analysis with standard sera. The obtained serological response was used as an indicator for success of the method. When coupling reactions were performed without the oxidation step no or almost no significant response of reference sera could be detected.
  • the immobilisation/coupling of the polysaccharide-protein complex to a carrier is such that high sensitivity and/or robustness is obtained.
  • flagellar antigens denature and lose their antigenicicty towards serum antibodies while the sensor chip has to be regenerated for a next analysis cycle with relatively harsh solvents
  • the somatic antigens are found rather stable towards these regeneration solvents.
  • the loss of immobilized O-antigen activity is believed to be primarily associated with degradation of the solid surface, namely gradual loss of dextran layer attached to the goldfilm, to which the antigens are bound.
  • the method according to the invention results in a carrier that is more robust compared to a carrier of the prior art.
  • the invention provides a method for immobilisation of a polysaccharide on a carrier, comprising contacting said polysaccharide with an oxidising agent and a protein to obtain a polysaccharide-protein complex and coupling said polysaccharide-protein complex to said carrier, wherein said polysaccharide is derived from a gram-negative bacterium and even more preferably wherein said polysaccharide is derived from an enterobacteriaceae. Yet even more preferably, said polysaccharide is derived from a gram-negative bacterium that is a human or veterinary or plant pathogen. Examples of such polysaccharides are polysaccharides derived from a salmonella (sub)species. Other examples are polysaccharides derived from Escherida coli species (for example E. coli 0157) and the bacterial species outlined in Table 2. Table 2. Examples of LPS-containing bacteria pathogenic to human and/or animals.
  • a carrier comprising an immobilised polysaccharide (O-antigen) is particularly useful in the diagnosis of the mentioned LPS-containing bacteria.
  • polysaccharide is intended to mean an entity comprising two or more glycoside linked monosaccharide units and embraces, amongst others, an oligosaccharide (2-10 residues) as well as a polysaccharide (more than 10 monosaccharides).
  • the linking may result in linear or branched polysaccharide.
  • the invention provides a method for immobilisation of a polysaccharide on a carrier, comprising contacting said polysaccharide with an oxidising agent and a protein to obtain a polysaccharide- protein complex and coupling said polysaccharide-protein complex to said carrier, wherein said polysaccharide is a lipopolysaccharide (LPS), i.e. a polysaccharide comprising lipid A.
  • LPS lipopolysaccharide
  • the used (lipo)polysaccharide must comprise at least one antigenic structure and one group available/suitable for providing a linkage between the protein and the polysaccharide. More details in respect of the last item will be provided later on.
  • the (lipo)polysaccharide comprises an antigenic structure and a group suitable for providing a linkage between the protein and the polysaccharide an immobilization method of the invention may be used to obtained a sensitive and/or robust carrier.
  • the LPS is expressed at the cellular exterior and is part of the bacterial cellular wall.
  • the expression of LPS is not under direct genetic control, so that LPS is a pool of different molecules with varying composition of the lipid A part in terms of the attached aliphatic chain.
  • the bacterial cell may synthesize rough LPS with no or a short carbohydrate chain, or smooth LPS with a mature carbohydrate chain existing of more than 50 repeating units expressing its antigenicity.
  • O-antigen profile is, however, per definition unique for a salmonella serogroup.
  • a complete serotyping of a salmonella also includes the H-antigens as well as the Vi-antigens.
  • LPS may be obtained by a variety of methods and the experimental part describes in more detail the use of a trichloric acid extraction (optionally followed by ethanol extraction and dialysis) according to Staub (1965) for this purpose.
  • suitable extraction methods are described by Wilkons (1996) and include, but are not restricted to, extractions with diethylene glycol, dimethyl sulphoxide, NaCl-diethyl ether (1:2 (v/v)), NaCl-butan-1-ol (1:1 (v/v)), aqueous EDTA, NaCl-sodium citrate, aqueous phenol or aqueous phenol- chloroform petroleum.
  • the purity of the obtained/used LPS batch is considered not to be extremely critical. It is experienced that the LPS does not have to be completely free of contaminants.
  • the specific coupling reaction provides a certain degree of selectivity.
  • the used/obtained LPS (preferably an LPS batch) is optimised in respect of the amount of protein necessary for an optimal response. It is clear to a skilled person that the LPS preferably comprises not much rough LPS.
  • the preferred LPS batch essentially comprises smooth LPS.
  • KDO and Hep and GIcNAc residues are KDO and Hep and GIcNAc residues.
  • the presence or absence of such a KDO and/or Hep and/or GIcNAc group is indirectly genetically determined.
  • the genetic information necessary to construct the species-, serotype or even strain specific monosaccharides is present in the corresponding organism, it depends on the growth circumstances whether said LPS contains aldehyde-convertible monosaccharides in the core region.
  • the invention provides a method for immobilisation of a polysaccharide on a carrier, comprising contacting said polysaccharide with an oxidising agent and a protein to obtain a polysaccharide- protein complex and coupling said polysaccharide-protein complex to said carrier, wherein said protein is a protein (for example a serum protein) with a certain amount of (primary) amines.
  • a protein for example a serum protein
  • at least some of these amines are not sterical hindered and/or are not participating in non-covalent bindings, such as H-H bridges or dipole-dipole interactions and/or are not protonated.
  • Such a protein preferably does not have or hardly have, any immunogenic properties and hence cross-reacting antibodies directed to the used protein are avoided as much as possible.
  • suitable proteins are haemoglobin (Hb), ovalbumin (Ob), myoglobin (Mb) and serum albumin (SA).
  • Hb haemoglobin
  • Ob ovalbumin
  • Mb myoglobin
  • SA serum albumin
  • the biosensor response of different standard sera on immobilised LPS oxidized in the presence of Hb or Ob or Mb or SA were determined. Serum albumin, myoglobin and haemoglobin gave the most promising results.
  • the protein is haemoglobin or myoglobin.
  • the necessary protein is obtained commercially or by (overexpressing) in a suitable expression system or by isolating it from a suitable source.
  • Haemoglobin has for example been obtained by isolating it from blood.
  • the used protein batches are as pure as possible, thereby circumventing as much cross-reactions as possible. It is however experienced that small amounts of contamination are allowed without jeopardising the sensitivity and/or robustness of the obtained carriers.
  • the ratio (lipo)polysachcharide versus protein depends, amongst others, on the used protein. Experiments with Hb have shown that concentrations between 15 and 50 % (m/m) have resulted in satisfactory results. When bovine serum albumin is used much lower ratios, between 0.7 and 7% (m/m), are used. Some examples: the optimal Hb concentration for S. livingstone LPS is around 50% (m/m) and for S. enteritidis LPS the optimal Hb concentration is 15% (m/m).
  • the isolated LPS preparations are preferably oxidised in the presence of a protein facilitated by an oxidising agent.
  • the invention provides a method for immobilisation of a polysaccharide on a carrier, comprising contacting said polysaccharide with an oxidising agent and a protein to obtain a polysaccharide-protein complex and coupling said polysaccharide- protein complex to said carrier, wherein said oxidising agent is capable of oxidising vicinal diols.
  • the oxidising agent preferably oxidises vicinal diols at least under controlled condition.
  • Oxidation of vicinal diols is preferred as this warrants reliable coupling of vicinal diol containing polysaccharide to the matrix.
  • the polysaccharides to be coupled to the matrix contain an antigen that is to be recognised by a member of a binding pair. To be recognizable it is preferred that the antigen is left unchanged at least in the majority of the polysaccharides that are being coupled to the carrier. This requires a balance between the level of oxidation required to obtain efficient coupling to the matrix and availability of the antigen for association with the member of the binding pair. The latter requires that the antigen is left essentially unaffected by the oxidation at least in an amount sufficient to be usable in a diagnostic setting.
  • Oxidation of vicinal diols according to the present invention warrants the availability of sufficient antigen in recognizable form while at the same time allowing efficient coupling of the polysaccharide to the carrier.
  • said oxidising agent comprises (sodium) m-periodate.
  • Other periodates such as potassium periodate or other salts thereof are also suitable periodates of the present invention.
  • Periodate oxidation is very suited for enabling preferential oxidation of vicinal diols according to the present invention. Oxidation of predominantly vicinal diols in a polysaccharide of the invention can typically be achieved by incubating said polyssaccharide with said periodate at a concentration of between 1 and 10 mM periodate.
  • incubation time When applying very short incubation times higher than 10 mM periodate can be used.
  • Periodate preferably oxidises vicinal diols, particularly of the more susceptible vicinal diols in the side chains of the polyssacharide.
  • vicinal diols will be oxidised.
  • a periodate oxidation is said to be mild when the mentioned preferred concentrations are used and when at least 20% and preferably at least 50%, more preferably at least 70% and most preferably about 90% of the antigen is intact after oxidation.
  • Availability or intactness of the antigen is preferably measured by means of an ELISA assay using a standardized antibody. Again we do not wish to be bound by any theory but it is currently thought that periodate will induce an oxidative disruption of linkages between vicinal diols on especially carbohydrate moieties, as in e.g. mannose, to yield aldehyde functionalities.
  • This reaction is typically performed in buffers at a pH range between 4.5 and 5.5 in the dark using a (preferably) freshly prepared 1-100 mM sodium meta-periodate in 0.1 M sodium acetate. Preferably the reaction is performed at a concentration of between 1 and 10 mM metaperiodate.
  • the oxidation is performed in the presence of a protein in the ranges as discussed above.
  • the bis-aldehyde compounds like the oxidised monosaccharide constituents in the polysaccharide chain of LPS, may react with any amino group in a protein and may form a Schiff-base linkage resulting in a substituted imine.
  • the inner core structure carries in most cases an oxidisable Gal, GIcNAc, Hep and/or KDO, but non-reducing Hep and KDO constituents are most susceptible for oxidation, in particular at very mild oxidation conditions at concentrations less than 6 mM meta periodate. Because the core region is a rather conserved part of LPS from different Enterobactericeae, (lipo)polysaccharides of members of the Enterobactericeae may be applied in a method of the invention.
  • Periodate will also oxidise, when present, certain aminoethanol derivatives such as the hydroxylysine residues in collagen, as well as methionine (to its sulfoxide) and certain thiols (usually to disulfides).
  • N- terminal serine and threonine residues of peptides and proteins can be selectively oxidized by periodate to aldehyde groups. These reactions, however, usually occur at a slower rate than oxidation of vicinal diols and the presence of such group does not substantially interfere with a method according to the invention.
  • the invention also provides a method for immobilisation of a polysaccharide on a carrier, comprising contacting said polysaccharide with an oxidising agent and a protein to obtain a polysaccharide-protein complex and coupling said polysaccharide-protein complex to said carrier, further comprising a step which results in ending/stopping the oxidation process, for example by desalting of said polysaccharide-protein complex.
  • a step which results in ending/stopping the oxidation process for example by desalting of said polysaccharide-protein complex.
  • This is for example accomplished with help of a NAP-5 column.
  • many other methods exist which have the same effect for example adding a reductor or an easily oxidisable molecule such as glycerol.
  • the way of stopping the oxidation is such that at the same time a buffer change is accomplished, for example HPLC, FPLC, dialysis, ion-exchangers, gel electrophoresis or ultrafiltration.
  • a buffer change for example HPLC, FPLC, dialysis, ion-exchangers, gel electrophoresis or ultrafiltration.
  • the invention therefore further comprises the obtained intermediate, i.e. the preparation of in the presence of protein oxidised polysaccharide, optionally desalted and optionally evaporated.
  • the used carrier is made of an inert, non-hydrophobic material and the binding of the LPS-protein complex to said carrier is covalent. Even more preferred such a carrier has a low protein binding or low biomolecular binding. Examples are a carrier of glass or silica or of a non-hydrophobic plastic.
  • said carrier is in the form of a microsphere or bead.
  • microsphere or beads are available to the person skilled in the art.
  • said microsphere or bead comprises polystyrene.
  • Microsphere or beads are particularly preferred because they can be provided with different antigens using a method of the invention. Microsphere or beads with different antigens can be accordingly coded with a different colour.
  • a sample for the presence of an antibody against an antigen can be done using a collection of the mentioned microsphere or beads. Binding of the antibody to a particular type of antigen can now be detected easily by the colour code of the microsphere or bead bound. Binding of the antibody can be detected in various ways. For instance, microsphere or beads containing bound antibody can be extracted from the sample and measured using a further antibody specific for the constant region of the antibody. On the other hand, sample can be directly analysed, i.e. in the absence of further manipulations by labelling the bound antibody and simultaneously detecting colour of the antibody and the colour of the microsphere or bead. Various methods for simultaneous detection of two or more colours are available to the person skilled in the art. In the present invention, a colour is defined as any type of electromagnetic radiation that can be detected, be it a typical colour revealed, for instance, by reflection of light, to light emitted as a result of fluorescence or phosphorescence.
  • the invention thus further provides a collection of at least two microsphere or beads wherein at least two of said at least two microsphere or beads each comprise a different antigen of the present invention.
  • said antigen comprises O-antigen of Salmonella.
  • said antigen is linked to said microsphere or beads carrier using a method of the invention.
  • At least one of said microsphere or beads comprises a polysaccharide coating linked to a polysaccharide comprising an antigen to be detected linked to each other via a polymer comprising at least two amine and/or amide groups, preferably a protein of the invention, wherein said linkage polymer (protein) is linked to said polysaccharide comprising said antigen, via an amine and/or amide group on said polymer and a periodate oxidised vicinal diol on said polysaccharide comprising said antigen.
  • the invention provides a method for immobilisation of a polysaccharide on a carrier, comprising contacting said polysaccharide with an oxidising agent and a protein to obtain a polysaccharide- protein complex and coupling said polysaccharide -protein complex to said carrier, further comprising activating the surface of said carrier.
  • said carrier comprises a glass surface coated with gold and even more preferred said carrier is modified with a carboxyl donor. A surface can be activated.
  • Carboxylic acid (COOH) groups (further referred to as carboxyl groups) are needed on this surface.
  • these COOH groups are provided by a stable homogeneous layer of molecules, which may have been modified for this purpose.
  • the carrier comprises a polysaccharide that acts as a carboxyl donor. More preferably a carboxymethylated dextran layer wherein said polysoaccharide modifed carrier, preferably comprising a dextran layer is activated with l-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide. The activation is preferably followed by preparation with carbohydrazine.
  • the polysaccharide-protein complex is added to the activated dextran layer.
  • the reactive aldehyde functionalities react spontaneously with the hydrazide to hydrazones, which are then reduced to stabilise the covalent bonds.
  • chip-conjugated LPS to bind anti-Enterobacterium (for example salmonella) antibodies is assessed using reference polyclonal agglutination sera.
  • serogroup-representing carbohydrates preferably 4 different serogroup-representing carbohydrates are used.
  • multiple (the amount of which is different on the particular question asked and on the used apparatus) different serogroup-representing carbohydrates are used and if one just wants to know whether for example an animal is or has been infected by a particular serogroup, a single serogroup-representing carbohydrate may be oxidised in the presence of a protein and immobilised on a carrier.
  • the use of at least two different serogroup-representing carbohydrates results in a carrier that can be used in a multi-serogroup analysis.
  • At least three and even more preferred at least more than three (for example four) different polysaccharides are used. These polysaccharides may be oxidised in the presence of one type of protein or in the presence of different types of protein.
  • the skilled person is capable of making any sensible combination. For example, to be able to detect more than 90% of all salmonella infections serogroups B, C and D in chicken and serogroups B, C, D and E in pigs should be represented.
  • Using one type of serogroup-representing carbohydrates is extremely useful if one is interested in the question whether or not a certain type of bacterium is or was present.
  • Using multiple different serogroup-representing carbohydrates is for example useful if one wants to determine whether an animal is or was infected by any gram-negative bacteria (for example enterob acteriace ae) .
  • the invention provides a method for immobilisation of a polysaccharide on a carrier, comprising contacting said polysaccharide with an oxidising agent and a protein to obtain a polysaccharide- protein complex and coupling said polysaccharide-protein complex to said carrier, wherein said carrier is a biosensor chip.
  • a biosensor chip is commercially available (for example that produced by Biacore) and hence no further information will be provided.
  • the invention provides a carrier obtained by the method according as described above or a carrier comprising an immobilised polysaccharide-protein complex on its surface.
  • a carrier of the invention comprises a polysaccharide coating that is linked to a further polysaccharide coating via reductive amination, wherein said further polysaccharide coating comprises a protein coupled to said further polysaccharide coating through oxidation of vicinal diols on said further polysaccharide - protein.
  • said reductive amination is achieved.
  • the invention provides a carrier comprising a polysacharide coating that is coupled to polysaachari.de
  • the invention provides biosensor comprising a carrier according to the invention.
  • Whether the carrier is obtained by a method according to the invention can for example be determined by extracting the polysaccharides from said carrier and determining whether covalently linked protein is present.
  • the carrier may also comprise different immobilised polysaccharides (for example O-antigens) possibly in combinations with different types of protein.
  • one type of protein may be used in the oxidation of different polysaccharides.
  • Whether a carrier and/or chip of the invention is employed can for example be determined with help of MALDI-MS possibly in combination with proteolytic digestion.
  • Such an analysis provides information with respect to the used protein and polysaccharide.
  • acidic hydrolysis the polysaccharide-protein complexes are released from the carrier.
  • LC-MS/MS analysis before and after proteolytic hydrolysis.
  • the obtained complex may also be subjected to a monosaccharide analysis, for example GC-MS following methanolysis and/or Smith degradation, from which it is determined which type of LPS is used. This information is furthermore used to determine whether KDO, Hep or other sugars have been oxidised.
  • a carrier of the invention may be used in different detection systems, for example optical, thermal, acoustic, amperometric, magnetic or chemical and a carrier of the invention may be used in any biomolecular interaction assay (BIA) or any affinity assay (AA).
  • BIOA biomolecular interaction assay
  • AA affinity assay
  • the invention provides a Surface Plasmon Resonance detection system comprising a biosensor as described above.
  • the gold layer in the sensor chip creates the physical conditions required for Surface Plasmon Resonance (SPR).
  • SPR Surface Plasmon Resonance
  • the principle of SPR will be described in the context of Biacore instruments. They incorporate the SPR phenomenon to monitor biomolecular interactions in 'real-time'. At an interface between two transparent media of different refractive index such as glass and water, light coming from the side of higher refractive index is partly reflected and partly refracted. Above a certain critical angle of incidence no light is refracted across the interface and total internal reflection (TIR) occurs at the metal film-liquid interface.
  • TIR total internal reflection
  • the electromagnetic field component termed the evanescent wave
  • the evanescent wave penetrates a distance on the order of one wavelength into the less optically dense medium.
  • the evanescent wave is generated at the interface between a glass prism (high refractive index) and a layer of buffer (lower refractive index). If the interface between the media of higher and lower refractive indices is coated with a thin metal film (a fraction of the light wavelength), then the propagation of the evanescent wave will interact with the electrons on the metal layer. Metals contain electron clouds at their surface, which can couple with incident light at certain angles.
  • a mass change of approximately 1 kRU (1,000 RU) corresponds to a mass change in surface protein concentration of 1 ng/mm 2 .
  • Typical responses for surface binding of proteins are of the order of 0.1-20 kRU.
  • the obtained carriers can be used in different types of analysis, such as bacteriology (direct assay) or serology (indirect assay).
  • An example of a serological assay is a method for determining the presence of an antibody directed to an antigen of a gram-negative bacteria in a sample, comprising contacting said sample with a carrier or a biosensor as described above and determining whether the carrier has bound any antibody ( Figure 1).
  • Such a method is for example very suitable for determining the presence of an antibody directed against an O antigen and thus it is indirectly established whether an infection is present or whether a recent infection has occurred.
  • Such a method is for example used to screen slaughter animals for salmonella or to screen animals for salmonella before they are exported abroad.
  • the method is also applied to samples obtained from living (for example, farm or zoo) animals.
  • samples that can be used in such a method are tissue sample, body fluid, secretes or excretes and more detailed examples are blood, blood derived samples, tissue, meat juice, milk, egg, fluids from an eye, saliva or faeces. As already outlined the samples can be obtained from dead as well as living animals.
  • a method according to the invention is not limited to a certain immunoglobulin (sub)type but can in principle be every (iso)type immunoglobulin such as (S)IgA 1 , (s)IgA2, IgD, IgG 1 , IgG2, IgG3, IgG 4 , IgM, IgY. Moreover, it may also be any other antigen-binding material. Preferably, such an antigen-binding material is a biomarker of a (history) of infection.
  • Such a serological assay is for example directed to one particular serogroup-representing carbohydrate or to different (i.e. multi analyte) serogroup-representing carbohydrates and hence such a method is for example used to determine the presence or absence of a certain salmonella (sub)type.
  • An example of a bacteriological assay is a method for determining the presence of a gram-negative bacterium in a sample, comprising contacting said sample with a predetermined amount of antibodies directed against an antigen of said bacterium and determining the amount of antibodies not bound to said bacterium with a carrier or a biosensor as described above.
  • the antigen is a serogroup-representing carbohydrate.
  • This method optionally further comprises the removal of non-bound antibodies from contacted sample and predetermined amount of antibodies by for example washing or immuno-magnetic separation procedures, centrifugation or filtering.
  • every type of sample can be used, such as animal feed, manure, feathers, soil, water for consumption or sewage water, meat, orange juice, chocolate, skin, vegetables etc.
  • Animal samples may be obtained from living as well as dead animals.
  • a single type of antibody as well as a mixture of at least two different types of antibodies directed against different antigens, for example two different serogroup-representing carbohydrates) is used.
  • such serological and bacteriological assays are performed such that the binding to said carrier or said biosensor is determined by Plasmon Surface Resonance or fluorescent microsphere or bead counter.
  • the source of the samples is as already outlined above unlimited and may for example be obtained from a human or an animal.
  • suitable animals are (race) horses, pigs, poultry (for example chicken, turkey, quail, duck, and goose), ruminants (for example calf or cow, goat, sheep).
  • the animals may be farm animals, zoo animals as well as free living animals.
  • samples from these animals may be obtained from living as well as dead animals.
  • the invention provides a method for determining the presence of a gram-negative bacterium in a sample comprising - contacting said sample with target bacteria-specific, bacteriophages and allowing the bacteriophages to infect said sample
  • the objective of this part of the invention is development of a fast (preferably within 24 h) and/or cost-effective and/or specific and/or sensitive diagnostic method for the determination of the presence of micro organisms. For this reason, the development of a biomolecular interaction assay (BIA) which exploits the ability of genus- and/or serovar-specific bacteriophages to multiply in their 'victim' bacteria, is aimed.
  • An increment in number of the target pathogen- specific phage(s) indicates not only the presence of the target organism but is also a (semi-)quantitative measure for the content of target bacteria in the tested sample.
  • a schematic overview of the proposed BIA method is depicted in Figure 2.
  • a particulate sample is homogenised for example using a Stomacher. Liquid samples are mixed by vigorous shaking. Analyte cells are then extracted or enriched by any suitable method and may comprise (a combination of) selective growth, centrifugation, filtration and/or immuno-magnetic separation (IMS). Enriched cells are fortified with target bacteria-specific bacteriophages and incubated for a few minutes while mixing. Before the multiplication cycle of the bacteriophage is complete, cells are washed to remove as complete as possible any non-bound and non-invading bacteriophages.
  • IMS immuno-magnetic separation
  • the sample is brought in contact with an indicator organism susceptible, i.e. in a life phase that is sensitive for bacteriophage penetration and intracellular multiplication, for the used bacteriophage, preferably at the highest possible concentration (for example concentrated overnight culture).
  • the bacteriophage-bacterium suspension is incubated for at least one bacteriophage multiplication cycle.
  • the phage-infected suspension is then centrifuged or filtered to precipitate/ remove cellular material and to recover multiplied bacteriophages.
  • the bacteriophage-containing sample is injected over an LPS-conjugated biosensor chip (according to the invention) to retain these particles in the detector for the generation of analyte-specific biosensor response.
  • the indicator organism can be kept as a continuous culture in the lab and has a cell density of usually 10 9 CFU/ml.
  • a suspension may be concentrated to 10 10 CFU/ml, as higher cell densities will increase sensitivity of the proposed method.
  • This method can be used to determine a single type of serovar but to detect multiple serovars in one run, a mix of different bacteriophages and a mixture of possibly different indicator bacteria may have to be applied.
  • Target bacteria-specific bacteriophages are described in the prior art and examples are provided in the experimental part, for example anti- Salmonella enteritidis bacteriophages.
  • Suitable carriers/chips are carriers/chips with LPS or with immobilised bacterial surface molecules (thus including membrane proteins and other biomolecules or a combination thereof).
  • LPS bacterial biomolecules
  • Use of LPS of cell membrane material will circumvent the generation of poly- or monoclonal antibodies. If attachment of the phages to bacterial biomolecules (LPS) is not satisfactory in the BIA, biosensor chip- immobilised anti-phage antibodies may have to be used in a successful BIA to capture bacteriophages from the probed sample.
  • the invention furthermore provides a kit with components suitable for use in any of the described applications.
  • such a kit comprises a ready -for use carrier/chip obtained by a method according to the invention.
  • the kit will at least comprise (lipo)polysaccharide fortified/enriched with protein (for example haemoglobin or serum albumin) in a predetermined amount, an amount of oxidizing agent (for example periodate), suitable buffers.
  • a kit comprises means for desalting, for example a desalting column.
  • the customer wants to mix (lipo)polysaccharide and protein himself these components are delivered separately together with an instructions manual.
  • such a kit may furthermore comprise positive and/or negative reference sera, a sample dilution buffer and any necessary instruction manual.
  • the methods as described above are particularly suitable for screening samples on a large-scale basis.
  • 96 samples were checked within 33 minutes.
  • a large-scale setting with relative slow biosensor equipment 15.000 samples were screened within 3 months. This number could have been much higher but unfortunately one of the slaughterhouses stopped participating.
  • Amine coupling kits consisting of iV-hydroxysuccinimide (NHS), l-ethyl-3-(3- dimethlylaminopropyl)carbodiimide hydrochloride (EDC) and ethanolamine hydrochloride — sodium hydroxide pH 8.5 and the running buffer (HBS-EP), containing 10 mM HEPES, 150 mM sodium hydrochloride, 3 mM EDTA and 0.005% (v/v) surfactant P20 at pH 7.4, were bought from Biacore AB (Uppsala, Sweden), which also supplied ready-to-use 10 mM glycine and 50 mM sodium hydroxide.
  • NHS iV-hydroxysuccinimide
  • EDC l-ethyl-3-(3- dimethlylaminopropyl)carbodiimide hydrochloride
  • HBS-EP running buffer
  • Ethanol, ethylene glycol, sodium chloride, sodium hydroxide and trichloroacetic acid (TCA) were purchased from Merck (Darmstadt, Germany).
  • Carboxymethylated-dextran sodium salt, sodium cyanoborohydride and carbohydrazide were obtained from Fluka Ghemie GmbH (Buchs, Switzerland).
  • CHAPS (Plus one) was delivered by Pharmacia Biotech (Uppsala, Sweden).
  • Sodium acetate trihydrate and acetic acid were supplied by J.T. Baker (Deventer, The Netherlands). Guanidine hydrochloride was obtained from Calbiochem (San Diego, CA, U.S.A.). Porcine haemoglobin (Hb) and myoglobin (Mb), chicken ovalbumin (Ob; 98% grade V), bovine serum albumin (BSA; 96% Fraction V), sodium periodate, Tween-20, Tween-80 and Triton X-100 were acquired from Calbiochem (San Diego, CA, U.S.A.). Porcine haemoglobin (Hb) and myoglobin (Mb), chicken ovalbumin (Ob; 98% grade V), bovine serum albumin (BSA; 96% Fraction V), sodium periodate, Tween-20, Tween-80 and Triton X-100 were acquired from Calbiochem (San Diego, CA, U.S.A.). Porcine haemoglobin (Hb) and myoglobin
  • NAP-5 columns (0.5 ml; Sephadex G-25) were purchased from Amersham Biosciences (Roosendaal, The Netherlands) and were used as described by the producer.
  • CM5 biosensor chips were bought from Biacore AB.
  • Dialysis bag (Spectra/Por) with a cut-off of 1 kDa was obtained from Spectrum Laboratories Inc. (Rancho Dominguez, CA, U.S.A.)
  • the following salmonella monovalent O' somatic lapine antisera were used: anti- 04, anti-O5, anti-06,7, anti-O8, anti-O9, anti-O10, anti-012, O Poly E (anti-O3, anti-O10, anti-O15, anti-019, anti-O34).
  • salmonella polyvalent O' somatic (PoIy A-S) lapine antisera (anti-O2, anti-03, anti-O4, anti-O5, anti-06,7, anti-O8, anti-O9, anti-O10, anti-Oil, anti-012, anti-013, anti-O15, anti-O16, anti-017, anti-018, anti-019, anti-O20, anti-021, anti-O22, anti-O23, anti-O28, anti-O30, anti-O34, anti-O35, anti-O38, anti-O40, anti-041) was used as well.
  • the sera were purchased from Pro-Lab diagnostics (Salmonella Reference Section of the Central Veterinary Laboratory, Weybridge, U.K.). Serogroup specific murine anti-B (anti-O4, 05 en 027), anti-C (anti-O7, 08), anti-D (anti 09, Vi) and anti-E (anti-O3, 019) monoclonal antibodies were bought from SIFIN (Berlin, Germany').
  • Sera were diluted 1:20 (v/v) in HBS-EP containing 1.0 M sodium chloride, 1% (m/v) carboxymethylated dextran and 0.05% (v/v) Tween 80, except anti-O5 serum was diluted 1:200 (v/v) and the anti- serogroup specific preparations were diluted 1:100 (v/v) in the same solvent.
  • C-Se, C-Spg and C-Si were diluted 1:200 (v/v) in HBS-EP containing 1.0 M sodium chloride, 1.0% (m/v) carboxymethylated dextran and 0.05% (v/v) Tween-80, whereas C-SPF and C-St were diluted 1:50 (v/v) in the same solution.
  • porcine sera from animals challenged with S. typhimurium and S. li ⁇ ingstone serogroup Ci
  • P-St and P-Sl porcine sera from animals challenged with S. typhimurium and S. li ⁇ ingstone
  • porcine serum used as control in a complement fixation test
  • porcine sera were diluted 1:20 (v/v) in HBS-EP containing 1.0 M sodium chloride, 1% (m/v) carboxymethylated dextran and 0.05% (v/v) Tween 80 as end concentrations.
  • Salmonella goldcoast (Sg; serogroup C2), S. li ⁇ ingstone (Sl) and S. melaegridis (Sm; serogroup Ei) were obtained from an in-house collection, while S. enteritidis #23 phage type Pt4 (Se), and S. typhimurium X- 193 phage type 507 (St) were kind gifts of F. G. van Zijderveld (Animal Sciences Group, LeIy stad, The Netherlands).
  • the bacteria were grown in overnight cultures in Nutrient Broth #2 (Oxoid, Basingstroke, U.K.).
  • a ⁇ -04 antibodies reacting with antigen structure coded with 04; in a similar way the antibodies against 05, 06,7, 09, OIO and 012 are indicated.
  • O antigens specific for salmonella serovar is indicated in brackets
  • Overnight cultures of salmonella were prepared by applying 100 ⁇ l from their corresponding stocks on each of the 120 plates containing brain heart infusion agar (BHIa, Oxoid). The presence of the expected salmonella serovar was confirmed through conventional selective growth, bio- and immunochemical classification, whenever new stock suspensions were produced.
  • the bacteria were harvested from the surface of the plates into 1 ml 9 g/1 NaCl (saline) solution per agar plate using a trigalski spatula. Each plate was washed twice with 2 ml saline solution. Bacteria were collected in six centrifugation tubes. Each tube was complemented with 100 ml saline and mixed before centrifugation at 10,000 g and 4 0 C for 15 min and supernatant was discarded.
  • This centrifugation step was repeated twice by suspending cells in 75 ml saline wash solution per tube each run. While kept on ice, pelleted bacteria were suspended in water at a volume ratio, which was a 5-fold to the weight of the bacteria. An equivolume of 0.250 M (Se) or 0.500 M (Sg, Sl, Sm and St) TCA was added to give end concentrations of 0.12 M and 0.25 M, respectively, followed by continuous stirring at 4 0 C for 3 h. A lipopolysaccharide (LPS)- containing supernatant was then acquired at 20,000 g and 4 0 C for 30 min.
  • LPS lipopolysaccharide
  • the pH of the supernatant was adjusted to pH 6.5 with 5 M sodium hydroxide and when nearing the aimed pH with 0.10 M sodium hydroxide.
  • the final volume of the LPS-containing solution was determined prior to storage at -18 0 C for 30 min.
  • the solution was diluted with a double volume of freezing cold absolute ethanol from a -18 0 C storage place, and incubation was continued overnight at -4 0 C without stirring in a closed, in house-build device with circulating cold ethylene glycol/water (1:4, v/v).
  • An LPS-containing pellet was obtained after centrifugation at 20,000 g and -4 0 C for 30 min.
  • the particulate material was suspended in a volume of 0.5 ml water per gram original bacterial mass weighed at the start of extraction process.
  • the suspension was dialyzed in a 1-kDa dialysis bag against water at 4 0 C for two days with regular intermittent refreshment of the water.
  • the bag content was centrifuged at 20,000 g and at 4 0 C for 30 min, and the supernatant was lyophilized.
  • the lyophilisate was weighed to establish the recovery of LPS.
  • LPS was reconstituted in water to make up an end concentration of 5 mg/ml.
  • Protein was added to an LPS preparation prior to its chemical modification and immobilization to a sensor chip to acquire high coating levels and high serum responsive antigens.
  • the optimum Hb content in each LPS batch was established by comparison of the responses of immobilized LPS that was fortified with Hb at different levels, using a panel of positive and negative reference sera.
  • a relative response indicative for a successful LPS immobilisation procedure is 2 kRU for a 62.5 ⁇ g/ml LPS solution containing 15% (m/m) protein, and 9 kRU for a 250 ⁇ g/ml LPS solution containing 50% (m/m) protein.
  • Optical SPR biosensor assays were performed on a Biacore 3000 SPR biosensor platform controlled by the same software as described above.
  • sera Prior to injection, sera were diluted in HBS-EP buffer containing 1.0% (m/v) carboxymethylated-dextran sodium salt, 1.0 M sodium chloride and 0.05% (m/v) Tween 80 at a ratio of 1:50 (v/v) or otherwise as indicated in the text.
  • the mixtures were incubated for at least 2 min at ambient temperature.
  • Pig sera were injected for 2 min at 40 ⁇ l/min, whereas bird sera were injected for 2 min at 5 ⁇ l/min or 20 ⁇ l/min as indicated.
  • Regeneration of the chip to recover the antigenic activity of the sensor surface was achieved with a 15-s pulse of 6 mM glycine at pH 2, containing 6 M guanidine hydrochloride, 0.1% (m/v) CHAPS, and 0.1% (v/v) of each Tween-20, Tween-80 and Triton X-100. This was followed with a second regeneration step with the running HBS-EP buffer enriched with 0.05% (m/v) CHAPS (end concentration) for 12 s at 100 ⁇ l/min.
  • Trimethylsilylated (methyl ester) methyl glycosides were prepared from the glycan samples by methanolysis (1.0 M methanolic HCl, 24 h, 85°C) followed by re-iV-acetylation and trimethylsilylation, and then analyzed by gas chromatography/mass spectrometry as described [Kamerling JP, Ventethart JFG (1989)].
  • the quantitative analysis was carried out by gas chromatography on a capillary EC-I column (30 m x 0.32 mm, Alltech) using a Chrompack CP 9002 gas chromatograph operated with a temperature program from 140°C to 240°C at 4°C/min, and flame-ionization detection.
  • the TCA concentration chosen as 'optimal' for LPS extraction from the different salmonella serotypes was based on the final LPS yields after dialysis, and were 0.12 M, 0.25 M, 0.25 M, 0.25 M and 0.25 M as end concentrations for Se, Sg, Sl, Sm and St, respectively.
  • pH of TCA-containing mixture is outlying ' some material was lost during sample work up process.
  • b batches Sm2003.1 to 2003.2 were combined to a single batch called Sm2003.1 Table 9.
  • the monosaccharide composition of isolated LPS preparations were analyzed to reveal the consistency of the isolation and purification procedure for LPS from different salmonella growths. It must be noted that analyses were performed on LPS preparations that were ready for oxidation and for that reason fortified with Hb at levels that were determined most optimal for the LPS batch tested (see below). For this purpose, GC-FID and GC-MS analyses were carried out after methanolysis of the Hb-fortified LPS preparations (Table 10 through Table 14). These results show that Hb does not contribute to a significant amount of carbohydrates in the final LPS preparation. Analysis of BHIa, showed the presence of exclusively galactose (Gal) and glucose (GIc).
  • GIcNAc can originate from either GIcNAc as in the repeating unit of Sl LPS, or from glucosamine (GIcN), which occurs as disaccharide in the lipid A moiety as backbone for the attached lipids.
  • GcN glucosamine
  • Gal occurs in the core region, which is conserved in all Salmonella enterica serovars, and in the PS region of Se, Sg, St and Sm as well. In these cases, the molar ratio of Gal is expected to be in excess of 1.0 Man, except for Sg in which each repeating unit contains 2 Man residues.
  • the monosaccharide analyses did not include the detection of O-acetylated, posphoryl-ethanolaminated or phosphorylated constituents, nor that of abequose (Abe; 3,6-dideoxy-xylohexose) or tyvelose (Tyv; 3,6-dideoxy-arabinose), which occur in the polysaccharide and core regions of the isolated LPS types as well.
  • the number of repeating units was estimated 19 and 20 in batches Se2003.1 and Se2003.4, respectively.
  • Nr of repeating 19 - - 20 units a does not include the contribution of Tyv residues; b Amount of LPS-containing material analysed was not accurately determined.
  • Nr of repeating units 9 8 a does not include the contribution of Abe residues.
  • the number of repeating units was calculated on the basis of the remaining core GIcNAc and Lipid A GIcN residues. This calculation revealed that the number of repeating units in Sl was also relatively small, namely 8 and 10 units in batch S12003.1 and batch2003.2, respectively.
  • Carbohydrate content 170 220 is Carbohydrate content 170 220
  • Nr of repeating units 12 13 a does not include the contribution of O-acetyl groups, which may be attached to the repeating Gal residues.
  • Batch St2003.2 contains less oxidizable Hep and KDO, which may affect the efficacy of the immobilization of LPS from this preparation.
  • Batch St2003.2 contains much more carbohydrate than batch St2003.1, namely 249 ⁇ g relative to 154 ⁇ g in 0.5 mg LPS, respectively.
  • Table 14 Monosaccharide analysis of S. typhimurium LPS. Batches St2003.1 and St2003.2 were fortified with Hb at 15% (m/m) and 25% (m/m), respectively. Molar ratios were determined on the basis of two GIcN and one GIcNAc residues (detected as three GIcNAc residues) present in the core and lipid A regions (referred to as CORE) and on the basis one Man residues in the repeating unit (referred to as UNIT). Normalized GIcNAc and Man residues are indicated by underlining. Carbohydrate content was determined in 0.5 mg LPS preparations.
  • Nr of repeating units 15 14 a does not include the contribution of (O-acetylated) Abe residues.
  • LPS did not couple through its KDO-carboxylic acid function to EDC/NHS-activated carboxymethated dextran, and no significant responses of reference sera were observed.
  • LPS was oxidized using sodium periodate to create reactive aldehyde groups in its carbohydrate constituents, which would allow the so-called aldehyde coupling procedure, i.e. condensation of aldehyde with a hydrazide function into a hydrazone linkage followed by reduction to a hydrazide product. Without oxidation, LPS had indeed little potential to immobilize to a surface of a CM5 chip coated with carbohydrazide (results not shown).
  • Biosensor responses following immobilization and responses of reference antisera flowed over chip surfaces, which were prepared with oxidized St LPS (batch St2003.1) in the presence of 7% (m/m) of the indicated protein.
  • Oxidation of LPS was necessary, as mixtures containing non-oxidized LPS and protein show relatively high immobilization levels but insignificant specific responses (Table 15).
  • protein addition was only beneficial prior to oxidation of LPS, as addition of BSA to oxidized and desalted St LPS gave acceptable immobilization levels but no expected serological responses (Table 16).
  • Hb yielded relatively high immobilization levels but no serological responses, as expected (results not shown).
  • proteins with relatively high degree of homology of their primary and secondary structure between homeothermic vertebrate species and occurring in serology-suitable matrices were selected for further investigations. For that reason, the performance of chicken ovalbumin, porcine haemoglobin, bovine serum albumin or porcine myoglobin fortified (7%, m/m) St LPS was compared (Table 16). Haemoglobin gave clearly best improvement of immobilization levels together with best expected antigen- antibody reactivity profile. In the presence of Hb, in particular, 012, poly O A-S and C-St reference sera gave better responses.
  • immobilization level did not correlate with serological responses but correlated with the amount Hb that was added.
  • Table 28 Responses of freshly oxidized Sg LPS (batch Sg2003.2) on indicated time points (in months).
  • the LPS was isolated from bacterial cells, fortified with 50% (m/m) Hb, dried and stored at 4-7°C until day of oxidation, immobilization and analysis.
  • Salmonella is one of the major causes of bacterial gastro-enteritis of humans (Fischer, 2004; van Duynhoven et al., 2005). In the Netherlands, between 1994-1998, Salmonella enterica serovar enteritidis (S.e.) was the most often isolated serovar (Pelt et al. 1999). Within this serovar, eggs and egg products were the most important source of infection. Despite several control measures, approximately 9 % of the Dutch layer flocks become infected annually. As egg contamination with Salmonella continues to be a threat for public health, it is important to detect an infection of a flock as soon as possible by an adequate surveillance programme.
  • the current Dutch monitoring system in layer finisher hens is based on serology (Bokkers, 2002).
  • the aim is to reduce the prevalence of S.e. and S. typhimurium in the layer sector. Sampling, however, occurs only twice: before and at the end of the laying period.
  • the current surveillance programme therefore, cannot detect all infections of flocks during the layer period, and farmers cannot 'guarantee' that their products are from Salmonella-free layers.
  • Egg sampling has the advantage that it can be performed on egg packing plants, in a high sampling frequency and with large sample sizes.
  • Tests for detection of antibody in eggs have been developed and used before.
  • the existing tests are often based on enzyme-linked immunosorbent assays (ELISA) using different (combinations of) antigenic components of Salmonella spp. (see for examples Refs. Gast et al, 2002 and 1997; Skov et al, 2002; Holt et al, 2000; Desmidt et ⁇ l., 1996; Gambweger et al, 1994; Van Zijderveld et al, 1992).
  • ELISA enzyme-linked immunosorbent assays
  • a biosensor consists of a re-usable immobilized biological ligand that 'senses' the analyte, and a physical transducer, which translates this phenomenon into an electronic signal (Jongerius-Gortemaker et al, 2002).
  • biosensors promises the possibility of high throughput analyses, and also the detection of multiple serovars or serogroups within a family of infectious disease agents -or antibodies against these agents- in a single run. This offers the opportunity to improve surveillance programmes, as more samples can be tested in a higher frequency during the layer period.
  • This example evaluates the sensitivity, specificity and discriminatory capacity of a surface plasmon resonance (SPR) biosensor (biacore 3000) antibody detection test in egg yolk based on the lipopolysaccharide (LPS) of Salmonella enterica sero ⁇ ar enteritidis and compares the results to those obtained with a.g,m fl ⁇ gellin based commercial ELISA test kit and a LPS based commercial ELISA test kit for detection of egg-antibodies by creating and analyzing receiver operating characteristic (ROC) curves.
  • SPR surface plasmon resonance
  • bias 3000 lipopolysaccharide
  • the egg yolk was diluted 1:5 (v/v) in 10 mM HEPES buffer at pH 7.4, containing 3 mM EDTA, 0.15 M sodium hydrochloride, 0.005 % (v/v) surfactant P20 (Biacore AB, Sweden), and additional 0.85 M sodium chloride (Merck, Darmstadt, Germany), 1 % (m/v) carboxymethylated dextran (Fluka Chemie, Buchs, Germany) and 0.05 % (v/v) Tween 80 (Merck, Germany). It was mixed with glass pearls, centrifuged at 15,000 g at ambient temperature for 25 min; the supernatant was filtrated over a 0.45- ⁇ m filter (Schleicher & Schuell, Dassel, Germany).
  • a second adaptation was the cleaning of the sensor chip. Following analysis of each series of 15 egg yolk samples, a solvent containing 0.5 % (w/v) sodium dodecyl sulphate (Biacore AB, Sweden) was injected to remove deposited egg yolk components.
  • a solvent containing 0.5 % (w/v) sodium dodecyl sulphate (Biacore AB, Sweden) was injected to remove deposited egg yolk components.
  • Sera were used as reference in the various tests due to unavailability of sample stock of well-defined reference egg yolks.
  • Lyophilized, defined SPF and reference sera originating from chickens infected with a) S. enteritidis, b) S. typhimurium, c) S. infantis, or d) S. pullorum were obtained from the Animal Health Service Ltd. (Deventer, Netherlands). These sera were prepared from pooled sera. Before use, lyophilized sera were reconstituted in 1 ml MiUi-Q. Additionally, monoclonal mouse anti- Salmonella antibody anti-group B, -group C, -group D and -group E was used (Sifin, Berlin, Germany).
  • Samples were assayed using sandwich enzyme immunoassay techniques. Two commercially available ⁇ S.e. antibody detection kits were used; Flockscreen S.e. Guildhay (Guildford, England) and FlockChek S.e. IDEXX (Westbrook, Maine, USA). The samples were analyzed according to the company's procedures.
  • the IDEXX S.e. competitive ELISA is based on g,m flagellar antigen. The wells of microtiter plates were coated with. g,m flagellar antigen, 1:2 dilutions of samples were added in mono. The results were expressed as S / N ratio as follows: optical density sample
  • the egg samples used in this study originated from two infection experiments.
  • Experiment 1 Fifteen one-week-old layer hens (Isa Brown) were housed in negative pressure high-efficiency particulate air filter (HEPA) isolators with a volume of 1.3 m 3 and fitted with a wire floor of 1.1 m 2 , and applying a 12 h light to 12 h dark photoperiod rhythm. The isolators were ventilated at a rate of approximately 30 m 3 /h. During the growing period, no Salmonella could be cultured from bedding. The chickens were provided with non-medicated feed and water ad libitum. They were housed, handled and treated following approval by the institutional animal experimental committee of the Dutch Animal Health Service Ltd.
  • HEPA high-efficiency particulate air filter
  • Eggs from experiment 1 were prepared in the following manner. To facilitate aseptic preparation, the eggshells were disinfected with a 70 % (v/v) aqueous ethanol. Subsequently, the eggs were cracked, and the contents were collected in sterile petri dishes. A volume of 1 ml egg yolk was collected using a sterile disposable syringe and portioned in 200 ⁇ l fractions. Each fraction was diluted with a buffer appropriate for either SPR biosensor or ELISA analysis, and then stored at -20 0 C.
  • pooled egg yolk and white and pre-ovulatory follicles obtained from experiment 2 were fractionated, diluted and stored at — 20 0 C.
  • SPR biosensor assay For this purpose, egg yolk was spiked with 1) S. enteritidis- (serogroups D), 2) S. infantis- (serogroups C), 3) S. pullorum- (serogroups D), and 4) S. typhimurium- (serogroups B) reacting antisera. These samples were diluted by their volumes either at a rate of 1:100 (1, 2 and 3) or at 1:50 (4). Further specificity testing was performed by spiking SPF egg yolk with 1:100 (v/v) diluted mouse monoclonal antibody reacting with Salmonella serogroups B, G, D and E.
  • the repeatability of the SPR biosensor assay was assessed by running the highly immuno-responsive egg yolk sample and the SPF negative control egg yolk sample twice on a single day and on three consecutive days (in triplo). Means, standard deviations (SD) and percent coefficient of variation (%CV) values were calculated in Excel 2000 (Microsoft software package).
  • Receiver operator characteristic (ROC) curves were generated using the results from the SPR biosensor and ELISA analyses to assess the test performances of each assay (Zweig and Campbell, 1993).
  • SPSS Receiver operator characteristic
  • AUC integrated area under the curve
  • SE standard error
  • SE probability of the null hypothesis of the true AUC being 0.5.
  • a ROC curve the true positive rate (sensitivity) is plotted in function of the false positive rate (100-specificity) for different cut-off points of a parameter.
  • Each point on the ROC curve represents a sensitivity/specificity pair corresponding to a particular decision threshold.
  • test signal of the SPF egg yolks spiked with S. enteritidis- (1:100, 145 RU), S. pullorum- (1:100, 1012 RU) or S. typhimurium- (1:50, 58 RU) positive sera were above the optimized cut-off value of 52 RU (cf. section 3.4.1 below) and considered positive, as was the SPF egg yolk spiked with mouse anti- Salmonella group D (1:100, 130 RU).
  • Non-spiked SPF yolk was found to be negative, i.e. average response was 30 RU.
  • the yolks spiked with S. infantis- (1:100, 24 RU) positive serum and mouse antiserum against Salmonella serogroups B (1:100, 27 RU), C (1:100, 16 RU), and E (1:100, 15 RU) were also below the cut-off value.
  • the coefficient of variation within a single day was 1 % for the highly immuno-responsive egg yolk sample and 13 % for the negative sample.
  • the coefficient of variation from day-to-day during three days was 2 % for the positive sample and 17 % for the negative sample.
  • ROG analysis was also performed for four combined egg white and yolk samples and 15 egg yolk samples from uninfected, and eight combined egg white and yolk samples and eight egg yolk samples infected chickens from experiment 2.
  • samples from the uninfected population gave biosensor responses ranging from 6 to 50 RU.
  • the responses of the samples from the infected population ranged from 11 to 3584 RU.
  • 24 out of 135 samples had to be considered immuno-negative.
  • a cut-off value of 52 RU yielded the highest possible diagnostic specificity estimate of 100 % (with a 95 % exact confidence interval (CI) of 95-100 %) and a diagnostic sensitivity estimate of 82 % (95 % CI: 76-98 %) for the SPR biosensor assay test.
  • a cut off value of 10 RU yielded the highest possible diagnostic sensitivity estimate of 100 % (95 % exact CI: 97-100 %) and a - specificity estimate of 1 % (95 % CI: 0-4 %).
  • a cut-off value of 42 RU yielded the optimal combined diagnostic sensitivity and -specificity: 84 % (95 % CI: 77- 90 %) and 99 % (95 % CI: 96-100 %), respectively.
  • the IDEXX ELISA had a diagnostic specificity of 100 % and a -sensitivity of 1 % (95 % CI: 0-3 %).
  • the OD 5 50nm of the samples from the uninfected population ranged from 0.174 to 1.377, i.e. in excess of the cut off value at 0.11.
  • 145 out of 147 samples had to considered immuno-negative at the chosen cut-off value, namely corresponding ODssonm ranged from 0.042 to 1.572.
  • ELISA had a diagnostic specificity of 100 % and a -sensitivity of 16 % (95 % CI: 10-22 %) at a cut-off value of OD ⁇ sonm 0.12. None of the samples from the uninfected population showed OD ⁇ sonm values in excess of 0.12 (0.051 to 0.093). In case of the positive population, 124 out of 147 samples had to be considered immuno-negative. The OD ⁇ sonm of these samples ranged from 0.048 to 1.471.
  • the aim of this study was to quantify the test characteristics of the SPR biosensor for the detection of S.e. antibodies in eggs.
  • the results showed that the SPR biosensor assay performed significantly better than the two commercially available ELISA's for samples from Experiment 1.
  • the combined optimal diagnostic sensitivity and -specificity of the SPR biosensor was 84 % (77-90 %) and 99 % (96-100 %), respectively.
  • Neither the g,m flagellin-b&sed IDEXX ELISA, nor the LPS-based Guildhay ELISA were able to detect S.e. infection with a higher combined diagnostic sensitivity and specificity using this test panel.
  • This study indicates that an SPR biosensor assay could be a new and powerful tool for monitoring Salmonella enterica serovar enteritidis infections in layer flocks through antibody detection in eggs.
  • the SPR biosensor assay offers the possibility of detecting infections in a fast and reliable way.
  • the high quality of the test and the technical and animal welfare advantages of egg collection are good reasons to explore its use for screening of populations.
  • the configuration of the applied SPR biosensor from Biacore allows the simultaneous detection of antibodies to multiple Salmonella serovars in a single run in a single sensor channel or in separate sensor channels on the same sensor chip (results not shown). This could be of significance because it is well known that serovars differ over countries and over time (see for examples Refs. Guerin et al., 2005; van Duijnkeren et al, 2002).
  • test evaluation was carried out using eggs from two experiments that were not carried out specifically for this test evaluation, possibly influencing test performance.
  • the 'positive' eggs were collected probably at a time point that humoral response was developing in the exposed chickens. These 'premature' eggs were analyzed and their false -negative results interfere with the evaluation of the assays.
  • the diagnostic sensitivity of each test, ELISA or SPR biosensor would have been improved.
  • Antibody detection in serum is more sensitive than in eggs, because the appearance of antibodies in eggs is preceded by the appearance in serum by a week (Gast and Beard, 1991; Sunwoo et al., 1996; Skov et al., 2002).
  • flock sensitivity of tests for antibodies in eggs can be improved by taking more samples, which is easier when using eggs.
  • the biosensor performance (AUC 0.892) was compared to that of two commercial ELISA's, (IDEXX AUC 0.432, Guildhay AUC 0.430). To our knowledge the IDEXX ELISA was not validated for eggs, but quantitative data exist about the test's performance in comparison to other tests: Van Zijderveld et al.
  • enteritidis represent 80% of isolates identified by the national reference laboratories participating in the Enter-net surveillance network between 1998-2003 (Fischer et al, 2004), this finding has limited clinical relevance for the human population.
  • the assay did differentiate between SPF egg yolk spiked with mouse anti-Salmonella group B (1:100, 27 RU) and D (1:100, 130 RU). This is not surprising, because the LPS of Salmonella enteritidis has O 1, O 9 and O 12 as somatic antigens, whilst the group specific test reagents contain the following monoclonal antibodies; anti-Salmonella group B: Anti-0 4, O 5, 027; anti- Salmonella group D: Anti-O9.
  • the cut-off value from Experiment 2 was much higher than the cut-off from Experiment 1, possibly because part of our samples consisted of egg white and yolk instead of egg yolk only. For different applications, different cut-off values may be optimal. Relative costs or undesirability of errors (false positive/false negative classifications) and the expected relative proportions of infected and uninfected hens are important parameters in the determination of the cut-off value, which affects the diagnostic value of the assay. We would suggest a cut-off value which minimizes the number of false positive results, reasoning that frequent sampling and testing would be necessary if the assay was to be used in a surveillance programme in the layer population, given the relatively low prevalence of S.e..
  • the SPR biosensor technique has successfully detected egg antibodies to determine experimental infections in chickens. In future screening programmes, the SPR biosensor could possibly detect different analytes at the same time.
  • Campylobacter is the most commonly food-borne pathogen in developed countries, causing gastroenteritis characterized by watery and/or bloody diarrhea. Campylobacter is associated with Guillain- Barre (GBS), Reiter's and haemolytic uremic (HUS) syndromes and reactive arthritis (FSAI, 2002; Lake et al., 2003; Tauxe, 2000). In the last 20 years, the infection rate of Campylobacter is still increasing in many developed countries, maybe due to the improvements in detection and reporting. In the United States of America, 2,400,000 cases of campylobacteriosis are reported annually corresponding to approximately 1% of the USA population (Tauxe, 2000).
  • Wild birds and domestic animals are reservoirs for Campylobacter and shed bacteria to the environment. Poultry is an importance vehicle for
  • Campylobacter is considered to be low, ranging from 500 — 10,000 cells (FSAI, 2002).
  • Campylobacter are Gram negative, curve, S-shaped, or spiral shaped bacilli having one or two flagella at one of the poles and highly motile (Ghristensen et al., 2001). Campylobacter grows between 30.5°C and 45 0 C at an optimum temperature of 42°C. Optimum growth is established at 10% carbon dioxide, 5-6% oxygen, and 85% nitrogen (FSAI, 2002).
  • IMS can potentially reduce pre- enrichment time of Campylobacter and may overcome the problems of inhibitors from food sources such as PCR inhibitors (Benoit and Donahue, 2003). The use of IMS may thus speed up the enrichment of the analyte.
  • This example describes a down-stream detection method using Campylobacter- specific bacteriophages, i.e. small viral organisms that attach to or infect living Campylobacter bacteria. Their attachment or infection is dependent of the phase of life cycle of the bacterium. Binding to or infection of Campylobacter may namely occur in the stationary, log or lag phase of the bacterium and depends of the phage species as well. Infection of the bacterium results usually in a high number of copies of the bacteriophage. Recording this increment of phages is therefore used as an analytical instrument to trace the presence of Campylobacter in the original sample.
  • Campylobacter- specific bacteriophages i.e. small viral organisms that attach to or infect living Campylobacter bacteria. Their attachment or infection is dependent of the phase of life cycle of the bacterium. Binding to or infection of Campylobacter may namely occur in the stationary, log or lag phase of the bacterium and depends of the phage species
  • the aim of this study is to demonstrate the application of bacteriophages as specific and sensitive analytical tools for the detection of Campylobacter in animal products, such as faeces and (poultry) meats.
  • IMS will be used to purify and concentrate Campylobacter from contaminated samples, such as meat and faeces.
  • IMS-isolated bacteria will be incubated with, an appropriate strain of bacteriophage. Non-attaching bacteriophages will be washed from the cell isolate using the same IMS procedure.
  • IMS-immobilised Campylobacter are then introduced in a fresh and pure culture of reference Campylobacter that is in a stationary phase. This cell culture is used as a foreign host to boost the multiplication of the bacteriophages.
  • bacteriophages will be harvested by centrifugation.
  • the bacteriophage-containing supernatant will be incubated with LPS-coated fluorescent beads.
  • the bead is coated as described in the Example with the LPS isolated from Campylobacter used as the host organism.
  • the presence of bacteriophages bound to the fluorescent beads will be tested in two ways. Following the addition of and incubation with anti- bacteriophage antibodies tagged with a fluorescent label, the amount of fluorescence will correspond with the concentration of bacteriophages and indirectly with the concentration of Campylobacter in the original sample. In an alternative approach, anti-LPS antibodies containing a fluorescent tag will compete with bacteriophages for binding places.
  • a decrease of recorded fluorescence compared to a Campylobacter-hee sample will, therefore, indicate a Campylobacter positive sample.
  • the test will be validated in terms of selectivity and sensitivity for C. jejuni, C. coli and C. larii in different matrices, including faeces, skin and meat from pigs and chickens. Closely related organisms, such as Arcobacter species, will be used to test the specificity of the method. Bacterial and viral strains and culture condition a jejuni (ATCC 33291) and C. coli (ATCC 33559) will be bought from Microbiologies (St. Cloud, USA).
  • the bacteria will grow in tryptone soya broth (TSB) (Oxoid, CM 129, Hampshire, England) for 24 h at 42°C, under microaerophilic atmosphere, which will be generated using a gas package (BBL, Becton Dickinson, Sparks, USA).
  • TLB tryptone soya broth
  • BBL Becton Dickinson, Sparks, USA
  • Campylobacter are then plated onto Charcoal-Cefoperazone-Deoxycholate Agar (mCGDA) (Campylobacter blood- free selective agar base [Oxoid, CM 739] with CCDA selective supplement [Oxoid, SR155], cefoperazone 32 ⁇ g/ml and amphotericin B 10 ⁇ g/ml) and incubated under microaerophilic atmosphere for 24 to 48 h at 42°C.
  • mCGDA Charcoal-Cefoperazone-Deoxycholate Agar
  • TSA tryptic soya agar
  • Campylobacter-infecting bacteriophages NTCC12669 , NTCC12670 , NTCC12671 , NTCC12672 , NTCC12673 , NTCC12674 , NTCC12675 , NTCC12676 , NTCC12677 , NTGC12678 , NTCC12679 , NTCC12680 , NTCC12681 , NTCC12682 , NTCC12683 , NTCC12684 are acquired from the National Type Culture Collection (London, United Kingdom).
  • Sample preparation The pure Campylobacter culture stored at 4°C will be subcultured in TSB and incubated under microaerophilic atmosphere for 24 h at 42°C. This is the host for exponential growth of the bacteriophage.
  • An amount of 25 g of ground chicken fillet will be suspended in 225 ml of Preston broth (Nutrient broth No.2 [Oxoid, CM 67], 5% (v/v) lysed horse blood [Oxoid, SR48], Campylobacter growth supplement [Oxoid, SR232] and modified Preston Campylobacter selective supplement [Oxoid, SR204]) contained by a stomacher bag.
  • the Preston broth medium will be prepared according to the manufacturer's instruction.
  • the sample-containing stomacher bag will be homogenized thoroughly for 90 s in a stomacher (Inter science, St.Nom, France). The entire suspension will be then be incubated under microaerophilic atmosphere at 42°C for an appropriate incubation time to allow growth of Campylobacter.
  • the stomacher bag containing the sample will be placed into the incubation pot of the IMS machine (PathatrixTM, Microscience, Cambridgeshire, UK). The apparatus is then operated according to the instructions of the manufacturer. Briefly, 50 ⁇ l of anti-Campylobacter magnetic beads (Microscience) will be added to the sample, which is then recirculated 30 min at 37°C.
  • the magnetically-immobilized beads are released, washed with 100 ml of pre-warmed buffered peptone water (peptone [Becton Dickinson] 10 mg/ml, sodium chloride [Merck, Darmstadt, Germany] 5 mg/ml, disodium hydrogen phosphate dihydrate [Merck] 4.5 mg/ml, potassium dihydrogen phosphate [Merck] 1.5 mg/ml adjusted to pH 7.2) and then drawn to the magnet again. Wash solution was removed leaving a 200 ⁇ l suspension for selective growth and bacteriophage analyses.
  • Campylobacter-carrying IMS-beads are contacted with a small volume of bacterium-specific bacteriophages. Following a short incubation to allow specific attachment of the phages to the surface of the targeted bacterium, IMS beads are washed and sampled to set a reference point in the final analysis procedure. The rest of the suspension is mixed with a suspension of fresh Campylobacter species to host the growing bacteriopage. Following incubation at 42°C, the suspension is centrifuged and the supernatant will be supplemented with a volume of Campylobacter LPS-coated fluorescent beads.
  • Multiplication of the phages is then assessed following the addition of either fluorescently labelled anti-bacteriophage antibodies or fluorescently labelled axiti-Campylobacter antibodies.
  • the beads are analysed using e.g. a BioPlex device (Bio-Rad) to screen fluorescence immobilised on the beads as a result of specific binding reactions.
  • Micro-organisms include a wide variety of bacteria, moulds (fungi), parasites and viruses. Pathogenic micro-organisms have attracted much attention from the public as consumers of contaminated food and water, which resulted in family or community outbreaks. As a consequence, the media and politicians have played their part in increasing consumer awareness and new legislation is in preparation or already in force.
  • zoonotic diseases i.e. microbes transmissible from animals to human, for the following reasons: 1) most food- and waterborne diseases in human are zoonotic by nature; 2) many zoonotic agents have their transmission route through the environment, and 3) both contamination of food/water and environment are also used by (bio)terrorists to acquire maximum impact in the society.
  • Microbiological hazards can enter food chains at any point during pre-harvest, production, processing, transport, retailing, domestic storage or meal preparation. From their introduction on feed or food, highly complex environments can occur in which the micro-organism can elude detection and inactivation. Efficient international distribution systems and rapid changes in consumer preferences can facilitate the swift penetration of pathogens through large populations, greatly shortening the reaction time available to public health agencies.
  • serology outperforms direct, and in most cases insensitive detection of tissue parasites, which can only be carried out by histochemistry or digestion techniques and microscopy.
  • Significant differences are also apparent in sample collection and preparation: whereas bacteria, fungi and viruses have to be cultured from matrices to facilitate their detection in enriched solutions, blood is relatively easily collected and prepared for analysis.
  • antibodies can not only be retrieved from blood, plasma or serum, but also from muscle (meat juice), milk, colostrums, cerebrospinal fluid and eggs.
  • sampling of eggs, meat juice and/or milk is easier and more cost-effective than the sampling of blood, plasma, serum or cerebrospinal fluid.
  • Salmonella serogroups are thus belonging to groups B, C and D, and in the case of swine also E.
  • Amine coupling kit consisting of iV-hydroxysuccinimide (NHS), l-ethyl-3-(3- dimethlylaminopropyl)carbodiimide hydrochloride (EDC) and ethanolamine hydrochloride — sodium hydroxide pH 8.5 were bought from Biacore AB (Uppsala, Sweden). Ethanol and trichloroacetic acid (TCA) were purchased from Merck (Darmstadt, Germany). Sodium cyanoborohydride and carbohydrazide were obtained from Fluka Chemie GmbH (Buchs, Switzerland). Porcine hemoglobin (Hb) was acquired from Sigma Chemical Company (St. Louis, MO, U.S.A.). Water was obtained from of a Milli Q water purification system (Millipore, Bedford, MA, U.S.A.). Materials
  • NAP-5 columns (0.5 ml; Sephadex G-25) were purchased from Amersham Biosciences and were used as described by the producer.
  • CM5 biosensor chips were bought from Biacore AB.
  • Dialysis bag (Spectra/Por) with a cut-off of 1 kDa was obtained from Spectrum Laboratories Inc. (Rancho Dominguez, CA, U.S.A.).
  • Alexa532 was from Molecular Probes (Leiden, The Netherlands).
  • Salmonella monovalent 'O' somatic monoclonal antisera against 04, 05, O6i, 07, 08, 09, OlO were purchased from Sifin (Berlin, Germany). Antibody solutions were diluted in 50 mM PBS to their working concentrations.
  • the obtained avian reference sera were reactive with Salmonella enteritidis (serogroup Di), S. typhimurium (serogroup B), S. pullorum I gallinarum (Spg; serogroup Di) and S. m/ ⁇ ni£s-(serogroup Ci), and were further referred to as CH-Se, CH-St, CH-Spg and CH-Si, respectively.
  • These chicken sera were originally prepared for ELISA analyses as positive references.
  • specific pathogen-free chicken serum was purchased from, this organisation as a negative control reference sample.
  • porcine sera from animals challenged with S. typhimurium and S. li ⁇ ingstone were acquired from GD and were referenced as P-St and P-Sl, respectively.
  • serogroup Ci porcine sera from animals challenged with S. typhimurium and S. li ⁇ ingstone
  • P-St and P-Sl were referenced as P-St and P-Sl, respectively.
  • Actinobacillus pleuropneumoniae serotype 2-reacting porcine serum (GD) used as control in a Complement Fixation Test was exploited as negative control for porcine serum in the salmonella biosensor assay.
  • Overnight cultures of salmonella were prepared by applying 100 ⁇ l from their corresponding stocks on each of the 120 plates containing brain heart infusion agar (BHIa, Oxoid).
  • the bacteria were harvested from the surface of the plates into 1 ml 9 g/1 NaCl (saline) solution per agar plate using a trigalski spatula. Each plate was washed twice with 2 ml saline solution. Combined bacteria were centrifuged in six tubes at 10,000 g and 4 0 C for 15 min and supernatant was discarded. This centrifugation step was repeated twice with 75 ml saline wash solution per tube each run.
  • pelleted bacteria were suspended in water at a volume ratio, which was a 5-fold to the weight of the bacteria.
  • An equivolume of 0.250 M (Se) or 0.500 M (Sg, Sl, Sm and St) TCA was added to give end concentrations of 0.12 M and 0.25 M, respectively, followed by continuous stirring at 4 0 C for 3 h.
  • a lipopolysaccharide (LPS) -containing supernatant was then acquired at 20,000 g and 4 0 C for 30 min. The pH of the supernatant was adjusted to pH 6.5 with 5 M sodium hydroxide and when nearing the aimed pH with 0.10 M sodium hydroxide.
  • the final volume of the LPS-containing solution was determined prior to storage at -18 0 C for 30 min.
  • the solution was diluted with a double volume of freezing cold absolute ethanol from a -18 0 C storage place, and incubation was continued overnight at -4 0 C without stirring in a closed, in house-build device with circulating cold ethylene glycol/water (1:4, v/v).
  • An LPS-containing pellet was obtained after centrifugation at 20,000 g and -4 0 C for 30 min.
  • the particulate material was suspended in a volume of 0.5 ml water per gram original bacterial mass weighed at the start of extraction process.
  • the suspension was dialyzed in a 1-kDa dialysis bag against water at 4 0 C for two days with regular intermittent refreshment of the water.
  • the bag content was centrifuged at 20,000 g and at 4 0 C for 30 min, and the supernatant was lyophilized.
  • the lyophilisate was weighed to establish the recovery of LPS.
  • LPS was reconstituted in water to make up an end concentration of 5 mg/ml.
  • Dependent of type of LPS and batch number a volume of 1 mg/ml porcine hemoglobine (Hb) was added to a concentration as indicated in the text.
  • Hb porcine hemoglobine
  • Oxidation O/LPS[MB7] A portion of 0.5 mg hemoglobin-fortified LPS was dissolved in 500 ⁇ l 100 mM sodium acetate pH 5.5. Following the addition of 20 ⁇ l 50 mM sodium periodate (Sigma), the solution was incubated for 40 min on ice protected from light. The oxidation of LPS was quenched and the solution was desalted by passing 500 ⁇ l of the reaction mixture through a NAP-5 cartridge with a gravity-controlled flow. Modified LPS was eluted with 1 ml 10 mM sodium acetate, pH 4.0. Prior to use, the cartridge was conditioned thrice with 3 ml 10 mM sodium acetate, pH 4.0.
  • the carboxylic groups at the bead surface were activated with a mixture of EDC/NHS available from the amine -coupling kit for 20 min on a gyro rocker. Following centrifugation and removal of supernatant, activation was followed by a reaction with 5 mM aqueous carbohydrazide for 20 min. Beads with modified surface were pelleted again and upper liquid was discarded before addition of 1 M ethanolamine and incubation for 20 min. Following another centrifugation step at 14,000 g for 5 min, oxidized LPS solved in sodium acetate pH 4.0 was added to allow immobilization for 90 min. Following removal of the suparenatant acquired through centrifugation, the linkage between bead-surface and antigen was stabilized using 100 mM sodium cyanoborohydride solved in 10 mM sodium acetate at pH 4.
  • this BioPlex (BioRad, Veenendaal, The Netherlands) was calibrated according to the instructions of the producer using a BioPlex calibration kit (BioRad).
  • Samples were diluted in 50 mM PBS in wells of a microtiter plate which were then supplemented with 50 ⁇ L 5000 beads/niL LPS-coated beads.
  • the antigen-antibody binding was allowed for 30 min on a microtiter plate shaker operated at 200 rpm.
  • 10 ⁇ L goat anti-swine IgG (H+L) tagged with Alexa532 fluorescent labels diluted 8 times in 50 mM PBS were added and incubation was continued for 15 min on the shaker. Beads were then analysed for their fluorescence profiles for 30 s on the BioPlex machine.
  • Fluorescent beads were prepared for coating with LPS from different specific Salmonella serovar sources representing serogroups B, C and D relevant as zoonoses in foods from chicken and swine. It should be noted that serogroup E, which is relevant for pork products, is not studied here. Following the immobilization of each type of LPS to individual beads, which are internally coded, the success of the coating was assessed using commercially available monoclonal antisera against somatic antigens 04, O5, O7, 08 and O9. However, while anti-O5 gave a response of 6398 units, anti-O9 gave 145 units, whereas the background signal of non-matching antigens-antibodies was less than 91 units in all cases (Fig. 6).
  • Serum from Salmonella- ⁇ ree pigs gave MFI responses in the range of 110 units (serogroup C2) to 137 units (serogroup B) and were close to the responses of beads only incubated with buffer, namely from 94 units (serogroup D) to 129 units (serogroup C2).
  • significant signals were recorded when sera were spiked with anti-S. typhimurium and & livingstone antisera, namely 969 units on serogroup B and 207 units on serogroup Ci, respectively.
  • the spiked sera did not react with non-corresponding antigens giving responses between 104 MFI units and 131 MFI units.
  • the aim of this investigation was to explore whether the developed SPR biosensor technology based on the use of immobilized selected Salmonella LPS to detect indirectly Salmonella infections in food-producing animals, is able to detect such infections in exotic animal species as well.
  • S. typhimurium phagetype 292 was isolated from the faeces of a Tocotoucan sampled at March 24, 1994. From another faeces sample of the same bird, S. typhimurium phagetype 352 was isolated at June 28, 1994. August 24,
  • the plasma prepared from this blood was used for analysis in this study. This animal died the next day. S. typhimurium phagetype 507 was isolated from the dead bird.
  • An SPR biosensor (Biacore 3000) containing a sensor chip of which flow channels were coated with LPS from S. enteritidis, S. li ⁇ ingstone, S. goldcoast and S. typhimurium, was operated as described earlier. Plasma samples were diluted as described for sera in examples 1 and 2 and analysed.
  • S. typhimurium as disclosed by classic microbiological diagnostics (personal communication with M. de Boer, Blijdorp Zoo, Rotterdam).
  • the faeces of the tocotoucan was found positive five and two months before blood was sampled and a humoral response could develop over a relatively long period of time. Indeed, the biosensor response was high (Table 33).
  • the reactivity with S. typhimurium LPS was dramatic high (4185 response units (RU)).
  • a response was also observed on the channel of S. enteritidis (1220 RU), which was also observed for serum from chickens highly infected with exclusively S.
  • Table 33 Results of the analysis of plasma collected from a tocotoucan and a sharp-tailed grouse which were infected with S. typhimurium. The results are expressed in relative response units. Samples were analysed thrice.
  • a dense network of ligands may be formed.
  • the density of the complex and the hindrance by the proteins may play a role in the observed difference in bacteriophage binding to the four LPS types.
  • bacteriophages propagated in Salmonella were concentrated, dialysed and serially diluted before SPR biosensor analysis (Fig. 17).
  • comparable bacteriophage concentrations gave dramatic different biosensor responses. Most probably phages were lost, in particular during concentration and dialysis step, during the sample preparation. Nevertheless, the preparations of bacteriophages, which have shown propogation (cf. Fig. 16) gave clearly higher responses than the blank sample, which contained the staring concentration of bacteriophages only.
  • bacteriophages can be used as an analytical tool to detect the presence of Salmonella in samples and that Salmonella LPS immobilized to a solid surface can be used to probe the increment of the bacteriophages as a result of propagation of the virus in the host bacteria following a short incubation period.
  • Lipid polysaccharides are potent immunogens, which can bring sensitive persons into a septic shock upon intake or inhalation. Precautions should be made to prevent contact with this biochemical.
  • Sodium periodate is an oxidizing agent and may cause explosions when brought in contact with strong reducing agents.
  • the procedure utilizes sodium cyanoborohydride.
  • the procedure should therefore be carried out with precautions as with for example hand gloves and a mask.
  • the material is very toxic to aquatic organisms and may cause long-term adverse effect in the aquatic environment.
  • the material and solution waste should be disposed of as hazardous waste.
  • somatic antigens are important as an instrument to trace immune response in animals upon an invasive infection of this micro-organism.
  • Somatic antigens are located on the polysaccharide part of lipid polysaccharide (LPS), which is a constituent of the bacterial cell wall.
  • LPS lipid polysaccharide
  • Salmonella serotypes c/. SOP CHEMIE/A21[AABI4]
  • a single species of beads can be immobilized with a mixture of LPS reflecting different serogroups, or different species of beads can be each immobilized with LPS reflecting a single specific serogroup or serovar of the pathogenic micro-organism.
  • the LPS-containing beads are incubated with body-derived materials, such as blood, plasma, serum, meat drip/juice, egg- yolk, milk etc , to enable anti- Salmonella antibody-antigen binding.
  • the specific binding is detected following a second incubation with fluorescently tagged antiimmunoglobulin antibodies in a device analyzing simultaneously the emission wavelengths of excitated beads and tagged antibodies. Only when both fluorescence of bead and antibody are detected simultaneously a response to a specific bead species will be recorded.
  • This SOP describes the method for oxidation, immobilization of LPS onto beads (Luminex) and an assay to assess the quality of produced.
  • LPS is oxidized in the presence of a protein facilitated by sodium periodate.
  • the LPS-protein solution is desalted using a NAP-5 column.
  • EDC l-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride
  • NHS -V-hydroxysuccinimide
  • desalted oxidized LPS-protein complex is immobilized to the solid phase of the beads.
  • Bound LPS is then stabilized with sodium cyanoborohydride.
  • the performance of bead-conjugated LPS to bind anti- Salmonella antibodies is assessed using a panel of reference monoclonal agglutination sera.
  • Periodate will induce an oxidative disruption of linkages between vicinal cis- diols on, in particular, carbohydrate moieties, as in e.g. mannose, to yield aldehyde functionalities, see Figure 18.
  • This reaction is typically performed in buffers at a pH range between 4.5 and 5.5 in the dark using a freshly prepared 10-100 mM sodium meta-periodate in 0.1 M sodium acetate.
  • Oxidation is performed in the presence of a protein.
  • the bis-aldehyde compounds such as the oxidized monosaccharide constituents in the polysaccharide chain of LPS here, may react with any amino group in a protein and may form a Schiff-base linkage resulting in a substituted imine. See Figure 19.
  • the carboxylie acid labeled beads are activated using -V-ethyl-A ⁇ -(3-dimethyl aminopropyl)-carbodiimide hydrochloride (EDC) and 2V-hydroxysuccinimide (NHS).
  • EDC -V-ethyl-A ⁇ -(3-dimethyl aminopropyl)-carbodiimide hydrochloride
  • NHS 2V-hydroxysuccinimide
  • the protein (R") carries multiple — NH2 groups and can therefore be conjugated with multiple oxidized LPS entities.
  • the polysaccharide part in LPS may carry multiple free aldehyde groups in a single molecule. These aldehyde groups may for a part or completely captured by the hydrazide-layer on the beads. The net result may be a very stable complex network of protein-LPS covalently linked to the bead surface.
  • KH2PO4 Potassium dihydrogen phosphate
  • Salmonella LPS in-house isolated LPS by TCA extraction (SOP CHEMIE/A21) prepared from the Salmonella bacteria serovars enteriditis (Se), goldcoast (Sg), livingstone (Sl), meleagridis (Sm) and typhimurium (St) with protein (SOP CHEMIE/A23) 8.1.10. Sheep anti-mouse Ig-PE (Chemicon, Boronia, Victoria, Australia)
  • Acetate buffer solution, c 10 mmol/1, pH 4.0: dissolve 0.272 g sodium acetate trihydrate (8.1.11) in 180 ml water and adjust to pH 4.0 with acetic acid (8.1.1) and make up to 200 ml with water. This buffer is stable for approximately six months.
  • Acetate buffer solution, c 1.0 mol/1, pH 5.5: dissolve 13.6 g sodium acetate trihydrate (8.1.11) in 90 ml water and adjust to pH 5.5 with acetic acid (8.1.1) and make up to 100 ml with water. This buffer is stable for approximately six months.
  • Acetate buffer solution, c 100 mmol/1, pH 5.5: dilute 1.0 ml acetate buffer solution, c ⁇ 1.0 mol/1 (8.2.3) with 9.0 ml water.
  • Carbohydrazide solution, c 100 mmol/1: dissolve 9.0 mg carbohydrazide (8.1.4) in 1000 ⁇ l water.
  • Carbohydrazide solution, c 5 mmol/1: dilute 10 ⁇ l carbohydrazide solution (8.2.5) with 190 ⁇ l water. Prepare just before use.
  • EDC-solution reconstitute EDC (8.1.2.2.) in 10.0 ml water.
  • NHS-solution reconstitute NHS (8.1.2.1) in 10.0 ml water.
  • PBS, c 10 mmol/1, pH 7.2: dilute 100 niL 10 times concentrated PBS (8.2.10) in 1 L water (8.1.17).
  • Anti-mouse Ig-PE, prediluted dilute fluorescent conjugate 5 times by mixing 40 ⁇ l anti-mouse Ig-PE (0) with 160 ⁇ l PBS (8.2.11).
  • the BioPlex apparatus is operated with Bio-Plex Manager software 4.1.
  • PROCEDURE 11.1. OXIDATION AND DESALTING OF LPS SOLUTION
  • Lipid polysaccharides are potent immunogens, which can bring sensitive persons into a septic shock upon intake or inhalation. Precautions should be made to prevent contact with this biochemical.
  • Sodium periodate is an oxidizing agent and may cause explosions when brought in contact with strong reducing agents.
  • the procedure utilizes sodium cyanoborohydride.
  • the procedure should therefore be carried out with precautions, such as using hand gloves and a mask. Use this substance only in a chemical fume hood.
  • the material is very toxic to aquatic organisms and may cause long- term adverse effect in the aquatic environment.
  • the material and solution waste should be disposed of as hazardous waste.
  • the LPS-containing solution (11.1.2.5) which can be used for different matrix and species applications is diluted as indicated in the following Tables 34 and 35.
  • Table 35 Amount of LPS solution used for the immobilization to fluorescent beads for detection of antibodies in chicken sera reacting with Salmonella O-antigens.
  • Beads (8.4.2) are vortex-mixed for minimally 1 min 11.2.2. Transfer a portion of 300 ⁇ L beads (11.2.1) into a fresh container (9.16) 11.2.3. Centrifuge at 14,000 g for 5 min
  • 11.2.28.7 Place the filled counting chamber under a microscope (9.12) and magnify the image with a 1Ox object. 11.2.28.8 The count should be started at the top left-hand corner and follow the direction shown by the arrow ( Figure 23, lower panel). Counting may be enhanced with the microscopes illumination reduced.
  • parynosides which are predominantly occurring in Salmonella LPS, need a higher periodate concentration to facilitate the oxidation in the same time.
  • Pyranosides which possess ⁇ -ery£/ ⁇ ro-hydroxyl groups, such as in arabino, galacto or manno configurations like in Salmonella spp. LPS, are easier oxidized than a-threo- hydroxyl groups, such as in xylo or gluco variants. It should be realized that while the ring is opened and aldehyde functions for attachment of e.g. protein molecules are created, also ⁇ -hydroxy carbonyl compounds may be created, which may oxidize again if periodate is still present.
  • LPS from Se, Sg, Sl and St was oxidized for 40 min at pH 5.5 using a range of sodium w-periodate concentrations. Following oxidation, LPS was coupled to a biosensor surface and immobilization efficiency and antigenic activities was monitored.
  • Salmonella spp. which are used in this extraction method to obtain lipopolysac ⁇ harides (LPS), belong to risk class 2 microorganisms.
  • Risk class 2 microorganisms are described as follows: they can induce illnesses like diarrhea and fever, but spreading of the disease is improbable and there are effective prophylaxes or treatment possible. The procedure when working with live bacteria should therefore be carried out with precautions like disinfecting equipment and hands, with 70% ethanol.
  • the waste of living Salmonella spp. should be destroyed or disposed of by autoclavation or as biohazard waste, respectively.
  • LPS compounds are highly pyrogenic and can cause fever. To avoid intake, treat aqueous solutions of LPS with care and wear a mask when working with solid material. If any of these compounds enters the bloodstream, immediately seek medical attention.
  • Salmonella is a gram-negative bacterium, and its outer membrane consists of various antigenic structures, including flagella, outer membrane proteins and Lipopoly saccharides (LPS).
  • LPS Lipopoly saccharides
  • the molecule of LPS consists of a so-called lipid A part, which is embedded in the leaflet of the outer membrane, a core region and polysaccharide.
  • the core region is composed of two or three heptoses and two or three residues of eight-carbon, negatively charged monosaccharides KDO.
  • the core region links lipid A to the polysaccharides, which is also known as the O-side chain. This O-side chain is highly variable with respect to its length and composition between strains, but also within a strain influenced by growth conditions of the Salmonella.
  • antigenic structures coded in the PS are unique for a certain Salmonella serovar.
  • antigenic structures 03, 04, O6/7, 08, 09, OIO and 012 represent approximately 90% of known Salmonella serovars occurring on porcine products, in particular, Dutch abattoirs.
  • LPS can be used to probe the binding of raised antibodies to these biomolecules.
  • LPS from S. typhimurium (04,05,012), S. enteritidis (09,012), S. li ⁇ ingstone (O6/O7), S. goldcoast (O6,O8) and S. meleagridis (O3,O10) can be extracted.
  • An in-house extraction is paramount because LPS from only a limited number of Salmonella serovars is commercially available. Furthermore, in-house production can secure a continuous availability of LPS types for a successful antibody detection assay.
  • the in-house extraction method described here for this purpose is based on a protocol described by Staub (O). Trichloroacetic acid (TCA) extracts LPS containing 1-10% protein contamination. This product is suitable for covalent immobilization of LPS to a carboxymethylated dextran layer coated on a gold metal surface of a biosensor chip (see SOP CHEMIE/A22 (Example 12)). This chip immobilized with LPS, in combination with a Biacore optical SPR biosensor system, can be used to trace Salmonella-LPS antibodies in sera also known as serology.
  • This method describes the extraction of LPS from several Salmonella serovars with the use of trichloric acetic acid. Extracted LPS is suitable for modifications to facilitate its immobilization on a earboxymethylated dextran surface.
  • SOP Chemie/A22 Immobilisation of Salmonella-derived LPS onto a biosensor chip (Biacore) and detection of serum antibodies reporting a current or past Salmonella infection (Example 12).
  • SOP Chemie/A23 Optimalisation of protein addition to LPS for immobilization and detection of serum antibodies (Example 13).
  • LPS Lipopolysaccharides
  • TCA trichloricacetic acid
  • Salmonella is cultured on and then collected from Brain Heart Infusion agar plates. After several washings steps with a saline solution and several centrifugation steps, TCA is added. The acidified suspension is incubated at a low temperature for three hours to solubilise LPS from bacterial cells. The suspension is centrifuged to remove cellular material and the pH is neutralized. LPS is then partly purified and concentrated by ethanol precipitation at low temperature. Finally, salts and ethanol are removed by dialysis, and remaining particles in the retained LPS- containing solution are spun down by centrifugation. The supernatant is lyophihzed and weighed to determine the recovery of produced LPS.
  • TCA trichloricacetic acid
  • BHI broth Dissolve 37 g of BHI broth (6.1.2) in 1.01 water (6.1.11). Mix well, distribute into final containers and sterilize by autoclaving at 121 0 C for 15 min.
  • BHIa Brain Heart Infusion Agar
  • NB Nutrient broth
  • TCA Trichloroacetic acid
  • TCA Trichloroacetic acid
  • LPS is a well-known toxin, which when inhalated or swallowed may induce clinical effects in humans.
  • Table 38 Conversion table to determine volumes of water (x),TCA (x), ethanol (e) and TCA concentration (y) for extraction and precipitation, and volume of water (z) for dialysis procedures to isolate and purify LPS from Salmonella. Letters noted: m refers to mass (9.3.23), whereas v refers to pH adjusted supernatant volume (9.3.34).
  • Lipid polysaccharides are potent immunogens, which can bring sensitive persons into a septic shock upon intake or inhalation. Precautions should be made to prevent contact with this biochemical.
  • Sodium periodate is an oxidizing agent and may cause explosions when brought in contact with strong reducing agents.
  • the procedure utilizes sodium cyanoborohydride.
  • the procedure should therefore be carried out with precautions as with for example hand gloves and a mask.
  • the material is very toxic to aquatic organisms and may cause long-term adverse effect in the aquatic environment.
  • the material and solution waste should be disposed of as hazardous waste.
  • Somatic antigens are important as an instrument to trace immune response in animals upon an invasive infection of this organism.
  • Somatic antigens are located on the polysaccharide part of lipid polysaccharide (LPS), which is a constituent of the bacterial cell wall.
  • LPS lipid polysaccharide
  • SOP CHEMIE/A21 Example H
  • antigen-containing LPS is coupled covalently to a biosensor chip surface to monitor serologically samples for the presence of anti- Salmonella antibodies through their binding to the immobilized antigen-containing LPS on the chip surface (see SOP CHEMIE/A23 (Example 13)).
  • SOP describes the method for oxidation, immobilization of LPS onto the biosensor chip (BIACORE) and the analysis of antibodies in sera.
  • LPS Sm from the Salmonella bacteria serovars meleagridis
  • LPS is oxidized in the presence of a protein facilitated by sodium periodate.
  • the LPS-protein solution is desalted using a Nx4P-5 column.
  • the LPS-protein complex is immobilized on a CM5-chip after activation of the carboxymethyl dextran layer on a biosensor chip with the aid of l-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride (EDC) and
  • Periodate will induce an oxidative disruption of linkages between vicinal diols on, in particular, carbohydrate moieties, as in e.g. mannose, to yield aldehyde functionalities.
  • This reaction is typically performed in buffers at a pH range between 4.5 and 5.5 in the dark using a freshly prepared 10-100 mM sodium meta-periodate in 0.1 M sodium acetate ( Figure 18).
  • Figure 18 The positions of conjugations indicated in this scheme, to link up this monosaccharide within a polysaccharide as e.g. LPS are just examples.
  • R' and Il indicate the distal and the proximal positions, respectively, in the carbohydrate chain.
  • the oxidation into aldehydes may repeat itself within the polysaccharide chain in each monosaccharide constituent containing vicinal diols.
  • Oxidation is performed in the presence of a protein.
  • the bis-aldehyde compounds like the oxidised monosaccharide constituents in the polysaccharide chain of LPS here above, may react with any amino group in a proteins and may form a Schiff-base linkage resulting in a substituted imine
  • the sensorchip -located carboxymethylated dextran layer is activated by N- ethyl-.-V-(3-dimethyl aminopropyl)- carbodiimide hydrochloride (EDC) and iV- hydroxysuccinimide (NHS).
  • EDC ethyl-.-V-(3-dimethyl aminopropyl)- carbodiimide hydrochloride
  • NHS iV- hydroxysuccinimide
  • the activation is followed by preparation with carbohydrazine.
  • the reactive aldehyde functionalities react spontaneously with the hydrazide to hydrazones, which are then reduced to stabilise the covalent bonds (Figure 34).
  • the protein R" carries multiple -NBfe groups and can therefore be conjugated with multiple oxidized LPS entities.
  • the polysaccharide part in LPS may carry multiple free aldehyde groups in a single molecule. These aldehyde groups may for a part or
  • Amine coupling kit (Biacore AB, Uppsala, Sweden) consisting of: 8.1.2.1. Vial containing 115 mg iV-hydroxysuccinimide (NHS)
  • Salmonella anti group specific, monoclonal test reagents 8.1.9.1. anti- Salmonella gr. B (SIFIN, Berlin, Germany), contains mAb Anti-04, 05, 027
  • anti- Salmonella gr. D contains mAb Anti-O9, Vi 8.1.9.4.
  • anti- Salmonella gr. E contains mAb Anti-O3, 019
  • Salmonella monovalent 'O' somatic anti sera 8.1.10. Salmonella monovalent 'O' somatic anti sera:

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Abstract

L'invention se rapporte au domaine de la chimie et du diagnostic, notamment au diagnostic d'infections présentes et/ou passée et/ou sans symptôme ou à un historique de l'exposition à une bactérie gram-négatif (telle qu'une enterobacteriaceae ou une legionella). L'invention porte encore plus particulièrement sur le dosage de la présence de micro-organismes indésirables chez des animaux ou des produits animaux. Elle concerne encore une méthode de dosage de la présence d'anticorps dirigés contre des micro-organismes indésirables dans des échantillons, méthode de préférence mise en oeuvre à l'aide d'un biocapteur. Une méthode d'immobilisation de polysaccharides sur des surfaces solides, des surfaces solides à polysaccharides immobilisés ainsi que les applications desdites surfaces sont décrites.
EP06733024A 2005-04-22 2006-04-24 Immobilisation de carbohydrates antigéniques pour contribuer à la détection de micro-organismes pathogènes Withdrawn EP1877794A1 (fr)

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WO2008067003A2 (fr) 2006-09-27 2008-06-05 Cmed Technologies Ltd. Procédé de détection des marqueurs immunologiques liés à un virus permettant de diagnostiquer des infections des voies respiratoires
US8110408B2 (en) 2006-09-28 2012-02-07 Cmed Technologies Ltd. Method for quantitative detection of diabetes related immunological markers
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